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Background and aims Messenger RNA (mRNA)-based therapeutics are rapidly progressing to the clinic and hold tremendous potential for benefiting millions of people worldwide. Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive aspect, however until recently, it has remained poorly explored. In this study, we examined for the first time, the therapeutic utility of mRNA delivery in liver fibrosis and cirrhosis, which contributes to millions of deaths, annually. Here, we aimed to demonstrate therapeutic efficacy of the human transcription factor hepatocyte nuclear factor alpha (HNF4A) encoding mRNA in chronically injured murine liver leading to fibrosis and cirrhosis. Methods We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNP) encapsulating HNF4A mRNA. To identify potential mechanisms, we performed microarray-based gene expression profiling, single cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. Results Expression of HNF4A encoding mRNA led to restoration of metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of HNF4A mRNA encapsulated-LNP induced a robust inhibition of fibrogenesis in four independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Conclusion Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. Lay summary Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality, worldwide. Herein, we take advantage of a promising, emerging mRNA therapy approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides the proof-of-concept that mRNA therapy would be a promising strategy for reversing liver fibrosis and cirrhosis.

Hepatocyte transplantation emerged a few decades ago as a possible strategy to overcome some of the limitations of liver transplantation, among them the availability of organ donors and the functional quality of grafts. Nowadays, allogenic cell transplantation is still to be confirmed as a viable therapeutic option for patients with hereditary metabolic liver diseases. Although we have now overseen 5 decades of basic and animal research in the field, the number of successfully treated patients remains low. Limitations in cell engraftment and selective proliferation of transplanted cells remain a constraint to the generalized use of this therapeutic approach.

Background & Aims Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The crosstalk between hepatocytes and hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis. Although ample efforts have been devoted to elucidate the functions of HSCs during liver fibrosis, the regulatory functions of hepatocytes remain elusive. Methods Using an unbiased functional microRNA (miRNA) screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via miRNA modulation. The in vivo functional analyses were performed by inhibiting miRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride- and 3,5-di-diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. Results Blocking miRNA-221-3p function in hepatocytes during chronic liver injury facilitated recovery of the liver and faster resolution of the deposited extracellular matrix. Furthermore, we demonstrate that reduced secretion of C–C motif chemokine ligand 2, as a result of post-transcriptional regulation of GNAI2 (G protein alpha inhibiting activity polypeptide 2) by miRNA-221-3p, mitigates liver fibrosis. Conclusions Collectively, miRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis. Lay summary Liver fibrosis majorly contributes to mortality resulting from various liver diseases. We discovered a small RNA known as miRNA-221-3p, whose downregulation in hepatocytes results in reduced liver fibrosis. Thus, inhibition of miRNA-221-3p may serve as one of the therapeutic approaches for treatment of liver fibrosis.

Background & Aims Current hepatic differentiation protocols for human embryonic stem cells (ESCs) require substantial improvements. MicroRNAs (miRNAs) have been reported to regulate hepatocyte cell fate during liver development, but their utility to improve hepatocyte differentiation from ESCs remains to be investigated. Therefore, our aim was to identify and to analyse hepatogenic miRNAs for their potential to improve hepatocyte differentiation from ESCs. Methods By miRNA profiling and in vitro screening, we identified miR-199a-5p among several potential hepatogenic miRNAs. Transplantation studies of miR-199a-5p-inhibited hepatocyte-like cells (HLCs) in the liver of immunodeficient fumarylacetoacetate hydrolase knockout mice ( Fah −/− /Rag2 −/− /Il2rg −/− ) were performed to assess their in vivo liver repopulation potential. For target determination, western blot and luciferase reporter assay were carried out. Results miRNA profiling revealed 20 conserved candidate hepatogenic miRNAs. By miRNA screening, only miR-199a-5p inhibition in HLCs was found to be able to enhance the in vitro hepatic differentiation of mouse as well as human ESCs. miR-199a-5p inhibition in human ESCs-derived HLCs enhanced their engraftment and repopulation capacity in the liver of Fah −/− /Rag2 −/− /Il2rg −/− mice. Furthermore, we identified SMARCA4 and MST1 as novel targets of miR-199a-5p that may contribute to the improved hepatocyte generation and in vivo liver repopulation. Conclusions Our findings demonstrate that miR-199a-5p inhibition in ES-derived HLCs leads to improved hepatocyte differentiation. Upon transplantation, HLCs were able to engraft and repopulate the liver of Fah −/− /Rag2 −/− /Il2rg −/− mice. Thus, our findings suggest that miRNA modulation may serve as a promising approach to generate more mature HLCs from stem cell sources for the treatment of liver diseases.

Stem cell transplantation has been proposed as an attractive alternative approach to restore liver mass and function. Recent progress has been reported on the generation of induced pluripotent stem (iPS) cells from somatic cells. Human-iPS cells can be differentiated towards the hepatic lineage which presents possibilities for improving research on diseases, drug development, tissue engineering, the development of bio-artificial livers, and a foundation for producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. This focused review will discuss how human iPS cell advances are likely to have an impact on hepatology.

In recent years the interest in liver cell therapy has been increasing continuously, since the demand for whole liver transplantations in human beings far outweighs the supply. From the clinical point of view, transplantation of hepatocytes or hepatocyte-like cells may represent an alternative to orthotopic liver transplants in acute liver failure, for the correction of genetic disorders resulting in metabolically deficient states, and for late stage liver disease such as cirrhosis. Although the concept of cell therapy for various diseases of the liver is widely accepted, the practical approach in humans often remains difficult. An international expert panel critically discussed the recent published data on clinical and experimental hepatocyte transplantation and the possible role of stem cells in liver tissue repair. This paper aims to summarise the present status of cell based therapies for liver diseases and to identify areas of future preclinical and clinical research.

hepatocytes and HepG2. The overexpression of p27 reduced cell proliferation and prevented AFB1-associated cell damage events in HepG2. The expression of p21, but not p27, was related to differentation staging of HCC. Conclusions: 1) AFB1 increases damage in DNA, reduces the expression of p27 and increases cell death in cultured hepatocytes. 2) The deficiency of p27 enhanced DNA damage and increased cell death in AFB1-treated hepatocytes. 3) The minor proliferative degree induced by overexpression of p27 was related to a reduction of DNA damage and cell death in AFB1treated HepG2 cells. 4) The expression of p21, but not p27, was associated with increased of differentiation of tumor in HCC.